• Inicio
  • Resultados de la búsqueda
  • A fast capillary electrophoresis method to assess the binding affinity of recombinant antithrombin toward heparin directly from cell culture supernatants.

A fast capillary electrophoresis method to assess the binding affinity of recombinant antithrombin toward heparin directly from cell culture supernatants.

Journal of pharmaceutical and biomedical analysis (2015-04-12)
Anne-Lise Marie, Nguyet Thuy Tran, Elsa P Bianchini, François Saller, Stéphane Pautus, Toufik Abache, Jean-Luc Plantier, Rémi Urbain, Delphine Borgel, Myriam Taverna
RESUMEN

With the aim to determine the binding affinity of a new generation of recombinant antithrombin (AT) toward heparin, we developed a dynamic equilibrium-affinity capillary electrophoresis (DE-ACE) method. This method allows the determination of an AT-heparin binding constant (Kd) directly from the cell culture supernatant used to produce the AT variants. Eight measurements per AT variant are sufficient to determine an accurate Kd (uncertainty ≤ 22%, regression coefficient ≥ 0.97), which is not significantly different from the value obtained from a higher number of measurements. Due to the relatively short time required to determine the Kd of one AT variant (2h), this method has the potential for being a low throughput screening method. The method was validated by analyzing five AT variants, whose Kd have been reported in the literature using fluorescence spectroscopy. Finally, the method was applied to estimate the Kd of one new AT variant and one AT conformer, a latent form, that exhibits a significant loss of affinity.

MATERIALES
Referencia del producto
Marca
Descripción del producto

Sigma-Aldrich
HEPES, ≥99.5% (titration)
Sigma-Aldrich
HEPES, BioPerformance Certified, ≥99.5% (titration), suitable for cell culture
Sigma-Aldrich
Sodium phosphate monobasic, ReagentPlus®, ≥99.0%
Sigma-Aldrich
Ammonium sulfate, for molecular biology, ≥99.0%
Sigma-Aldrich
Sodium hydroxide solution, BioUltra, for molecular biology, 10 M in H2O
Sigma-Aldrich
Sodium hydroxide solution, 1.0 N, BioReagent, suitable for cell culture
Sigma-Aldrich
Thiourea, ACS reagent, ≥99.0%
Sigma-Aldrich
HEPES buffer solution, 1 M in H2O
Sigma-Aldrich
Selenium, powder, −100 mesh, 99.99% trace metals basis
Sigma-Aldrich
Sodium phosphate monobasic, BioReagent, for molecular biology, anhydrous, ≥98%
SAFC
HEPES
Sigma-Aldrich
Thiourea, ReagentPlus®, ≥99.0%
Sigma-Aldrich
Sodium phosphate monobasic, BioXtra, ≥99.0%
Sigma-Aldrich
Selenium, powder, −100 mesh, ≥99.5% trace metals basis
Sigma-Aldrich
HEPES, BioUltra, for molecular biology, ≥99.5% (T)
Sigma-Aldrich
Ammonium sulfate, BioXtra, ≥99.0%
Sigma-Aldrich
Sodium phosphate monobasic, BioPerformance Certified, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.0% (titration)
Sigma-Aldrich
Sodium phosphate monobasic solution, BioUltra, 5 M in H2O
Sigma-Aldrich
Sodium phosphate monobasic, meets USP testing specifications, anhydrous
Sigma-Aldrich
Sodium phosphate monobasic, anhydrous, free-flowing, Redi-Dri, ≥99.0%
SAFC
HEPES
Sigma-Aldrich
HEPES, BioXtra, pH 5.0-6.5 (1 M in H2O), ≥99.5% (titration)
Sigma-Aldrich
Ammonium sulfate, BioUltra, ≥99.0% (T)
Sigma-Aldrich
HEPES, BioXtra, suitable for mouse embryo cell culture, ≥99.5% (titration)
Sigma-Aldrich
Sodium hydroxide, BioUltra, for luminescence, ≥98.0% (T), pellets
Sigma-Aldrich
Sodium hydroxide, ultra dry, powder or crystals, 99.99% trace metals basis
Sigma-Aldrich
Ammonium sulfate, 99.999% trace metals basis
Sigma-Aldrich
Selenium, pellets, <5 mm, ≥99.99% trace metals basis
Sigma-Aldrich
Selenium, pellets, <5 mm particle size, ≥99.999% trace metals basis
Sigma-Aldrich
HEPES, anhydrous, free-flowing, Redi-Dri, ≥99.5%